SU575353A1 - Method of obtaining carboxypepiidase - Google Patents

Description

one

The invention relates to a method for producing a proteolytic fermoite carboxypeptindaea (EC 3.4.12) used in food, textile and medical washing, as well as in scientific and laboratory practice.

On an industrial scale, carboxypeptidae is obtained from animal raw materials - the bovine pancreas - by multistage purification and separation of the fraction containing carboxypeptidase. The result is a suspension of the enzyme, very unstable during storage.

Currently, the most promising O4I are methods for producing enzymes from microbial sources of cheap raw materials and a powerful producer of enzymes with specific properties.

One such method is to obtain the carboxypeptidase fraction from the pronase – enzyme complex of Streptomyce & grisetis (Str.) Actinomycete by precipitating proteins with salts, for example dimethyl sulphate, and organic solvents, fractioning proteins on ion exchange resins and molecular sieves with separation of the mixture enzymes that are part of

pronase, and kx chromatography on an amberlite column.

The protein of the carboxypeptidase fraction is precipitated from the solution by sulphate of akuni, the precipitate is dissolved in a buffer and then dialyzed to obtain fermitic in the form of a solution.

The disadvantages of this method include the low yield of the target product and the production of a fermitite with iekimi | specific activity and storage stability.

The purpose of the invention is to increase the stability of the enzyme, its specific activity and yield - by tTO from the culture fluid of actin midin Str. After filtration, the fHeeus precipitates the proteins by adding ammonium sulfate until complete saturation, and the carboxypepididase obtained after dialysis is lyophilized in acetate buffer containing calcium ions, preferably in 0.05 M acetate buffer containing 0.01 M calcium acetate

Claims (2)

Example. 1.5 l of culture liquid liquid octomycete Str. seus strain C-5 obtained from the Kiev medical plant, filtered, ammonium sulphate was added to the filtrate until complete saturation (700-720 g of salt per 1 L of filter), centrifuged (4000-6000 rpm in cold for 1 hour and the precipitate is suspended in 50 ml of water containing 0.001 M calcium acetate. Then the mixture is dialyzed for 4-5 hours with THS running water and 12 hours against distilled water with 0 | OQ1 M acetate per kg. The volume of the fluid after dialysis is increased to 100 ml Dialysate is applied to a {5 x X 40 cm column) I filled with sodium carboxy methylcellulose with a capacity 0.7 mg-ec Protein fractions are eluted with a linear gradient of sodium chloride (0.005 M acetate buffer containing 0.001 AND calcium ions in one vessel, and another 500 ml of a 0.35 M solution of sodium chloride in the buffer). The fraction obtained after chromatography with hydrogen is precipitated with a saturated solution (98%) of ammonium sulfate, centrifuged in the cold, dissolved in a minimum amount of 0.05 M acetate buffer containing 0.01 M calcium calcium, and dialyzed 2 hours against running water 12 hour vs buffer. The resulting dialysate is applied to a column (2 x 30 cm) with amberlite SU-50 in Ma form, equilibrated with the same buffer, and protein fractions are eluted with a linear sodium acetate gradient (in one vessel 500 ml 0.05 M acetate buffer containing 0, 01 M calcium ions, in another 1 M sodium acetate solution in the same buffer). The third fraction containing carboxypeptidase obtained after chromatography is precipitated with ammonium sulfate, dialyzed against acetate buffer, and lyophilized in 0.05 M acetate buffer containing 0.01 M calcium acetate. A carboxne peptidag preparation is obtained, homogeneous with rechromatography on a column with amberlite SU-50, with electrophoresis in polyacrylamide gel and in the N-terminal analysis (alanine). The advantages of the proposed method include the preparation of a highly active, stable (lyophilized preparation containing 5-10% protein, retains its activity for several years) of a carboxy-: peptidase preparation with a high yield (protein yield from culture protein 4- 5%, by activity 10-20%) from microbial raw materials, which is a waste in the production of antibiotics. Claim 1. Method for producing a carboxypeptide, protein from actinomycete Straptomvces protein raw material, including choros, extraction of proteins from raw material by ammonium sulfate precipitation, fractionation of proteins by ion-exchange chromatography with subsequent dialysis, characterized by its specific activity. and yield, as a protein raw material, actinomycete culture fluid is used, from which, after filtration, proteins are precipitated by adding ammonium sulfate to complete saturation, and the resulting after dialysis carboxypeptidase is lyophilized in acetated buffer containing calcium supplements. 2. Method POP1, characterized in that the lyophilization is carried out in 0.05 M acetate buffer containing 0.01 M calcium acetate.